Shallow Chemoattractant Gradients Research Articles

Sphingomyelin metabolism underlies Ras excitability for efficient cell migration and chemotaxis.

In eukaryotic motile cells, the active Ras (Ras-GTP)-enriched domain is generated in an asymmetric manner on the cell membrane through the excitable dynamics of an intracellular signaling network. This asymmetric Ras signaling regulates pseudopod formation for both spontaneous random migration and chemoattractant-induced directional migration. While membrane lipids, such as sphingomyelin and phosphatidylserine, contribute to Ras signaling in various cell types, whether they are involved in the Ras excitability for cell motility is unknown. Here we report that functional Ras excitability requires the normal metabolism of sphingomyelin for efficient cell motility and chemotaxis. The pharmacological blockade of sphingomyelin metabolism by an acid-sphingomyelinase inhibitor, fendiline, and other inhibitors suppressed the excitable generation of the stable Ras-GTP-enriched domain. The suppressed excitability failed to invoke enough basal motility to achieve directed migration under shallow chemoattractant gradients. The fendiline-induced defects in Ras excitability, motility and stimulation-elicited directionality were due to an accumulation of sphingomyelin on the membrane, which could be recovered by exogenous sphingomyelinase or phosphatidylserine without changing the expression of Ras. These results indicate a novel regulatory mechanism of the excitable system by membrane lipids, in which sphingomyelin metabolism provides a membrane environment to ensure Ras excitation for efficient cellular motility and chemotaxis.Key words: cell polarity, cell migration, Ras, excitability, sphingomyelin.

Two Complementary Signaling Pathways Depict Eukaryotic Chemotaxis: A Mechanochemical Coupling Model.

Many eukaryotic cells, including neutrophils and Dictyostelium cells, are able to undergo correlated random migration in the absence of directional cues while reacting to shallow gradients of chemoattractants with exquisite precision. Although progress has been made with regard to molecular identities, it remains elusive how molecular mechanics are integrated with cell mechanics to initiate and manipulate cell motility. Here, we propose a two dimensional (2D) cell migration model wherein a multilayered dynamic seesaw mechanism is accompanied by a mechanical strain-based inhibition mechanism. In biology, these two mechanisms can be mapped onto the biochemical feedback between phosphoinositides (PIs) and Rho GTPase and the mechanical interplay between filamin A (FLNa) and FilGAP. Cell migration and the accompanying morphological changes are demonstrated in numerical simulations using a particle-spring model, and the diffusion in the cell membrane are simulations using a one dimensional (1D) finite differences method (FDM). The fine balance established between endogenous signaling and a mechanically governed inactivation scheme ensures the endogenous cycle of self-organizing pseudopods, accounting for the correlated random migration. Furthermore, this model cell manifests directional and adaptable responses to shallow graded signaling, depending on the overwhelming effect of the graded stimuli guidance on strain-based inhibition. Finally, the model cell becomes trapped within an obstacle-ridden spatial region, manifesting a shuttle run for local explorations and can chemotactically “escape”, illustrating again the balance required in the complementary signaling pathways.

Multi-State Transition Kinetics of Intracellular Signaling Molecules by Single-Molecule Imaging Analysis.

The chemotactic signaling of eukaryotic cells is based on a chain of interactions between signaling molecules diffusing on the cell membrane and those shuttling between the membrane and cytoplasm. In this chapter, we describe methods to quantify lateral diffusion and reaction kinetics on the cell membrane. By the direct visualization and statistic analyses of molecular Brownian movement achieved by single-molecule imaging techniques, multiple states of membrane-bound molecules are successfully revealed with state transition kinetics. Using PTEN, a phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) 3'-phosphatase, in Dictyostelium discoideum undergoing chemotaxis as a model, each process of the analysis is described in detail. The identified multiple state kinetics provides an essential clue to elucidating the molecular mechanism of chemoattractant-induced dynamic redistribution of the signaling molecule asymmetrically on the cell membrane. Quantitative parameters for molecular reactions and diffusion complement a conventional view of the chemotactic signaling system, where changing a static network of molecules connected by causal relationships into a spatiotemporally dynamic one permits a mathematical description of stochastic migration of the cell along a shallow chemoattractant gradient.

Evolutionarily Conserved Coupling of Adaptive and Excitable Networks Mediates Eukaryotic Chemotaxis

Numerous models have been proposed to explain the remarkable ability of chemotactic cells to sense and migrate toward extremely shallow chemoattractant gradients independently of the ambient concentration. We carried out experiments to distinguish the various models of gradient sensing in migrating cells. First, signaling activity was strongly suppressed toward the low side of cells in a gradient or following sudden removal of uniform chemoattractant. Second, signaling activities displayed a rapid shut off and, with stimulation of increasing duration, a slower adaptation during which responsiveness to subsequent test stimuli declined. Simulations of existing classes of models indicated that these observations can only be explained by the coupling between an adaptive module and an excitable network. Moreover, stimulation of cells lacking G-protein function suppresses downstream activities, while constitutive G-protein activation induced persistent responses. This indicates that chemoattractant sensing is mediated by a G-protein-dependent excitor and a G-protein-independent inhibitor forming an incoherent feedforward loop. The salient features of the coupling between adaptive and excitable networks were observed for the chemoattractants cAMP and folic acid in Dictyostelium as well as fMLP in human neutrophils, suggesting an evolutionarily conserved mechanism for eukaryotic chemotaxis.

Evolutionarily conserved coupling of adaptive and excitable networks mediates eukaryotic chemotaxis.

Numerous models explain how cells sense and migrate toward shallow chemoattractant gradients. Studies show that an excitable signal transduction network acts as a pacemaker that controls the cytoskeleton to drive motility. Here we show that this network is required to link stimuli to actin polymerization and chemotactic motility and we distinguish the various models of chemotaxis. First, signaling activity is suppressed toward the low side in a gradient or following removal of uniform chemoattractant. Second, signaling activities display a rapid shut off and a slower adaptation during which responsiveness to subsequent test stimuli decline. Simulations of various models indicate that these properties require coupled adaptive and excitable networks. Adaptation involves a G-protein independent inhibitor since stimulation of cells lacking G-protein function suppresses basal activities. The salient features of the coupled networks were observed for different chemoattractants in Dictyostelium and in human neutrophils, suggesting an evolutionarily conserved mechanism for eukaryotic chemotaxis.

Bidirectional molecular transport shapes cell polarization in a two-dimensional model of eukaryotic chemotaxis

Chemotacting eukaryotic cells can sense shallow gradients of chemoattractants and respond by assuming an asymmetric shape with well-defined front and back regions. Such a striking polarization phenomenon is produced largely through the interconversions and interactions between several cellular components, including Rac GTPase (Rac), phosphoinositide 3-kinase (PI3K), tensin homology protein (PTEN), phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) and phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2). Here, we developed a mathematical model of cell polarization by exploring bidirectional molecular transport that arose from phosphoinositides (PIs) and Rac-mediated feedback loops. We assumed a static gradient of activated Rac derived from an external signal field as the internal trigger signal. The evolution of PI(3,4,5)P3 and PI(4,5)P2 along with PI3K and PTEN that act as activator and inhibitor, respectively, were described by a pair of coupled transient reaction-diffusion equations. The entire system was solved using a Lattice–Boltzmann method with an embedded Monte-Carlo method to track the stochastic translocation behaviors of discrete PI3K/PTEN molecules. We first showed that, upon a graded external stimulus, the Rac to PI(3,4,5)P3 cascade exhibited a short range positive-feedback loop, while the PTEN to PI(4,5)P2 cascade contributed another long range negative-feedback loop, which dominated the “forward” and “backward” molecular transport, respectively. Second, polarization was governed by the ratio of [PI3K] to [PTEN], and manifested a switch-like behavior. Third, with a uniform stimulus, spontaneous polarization could occur in PTEN-deficient cells.

Intracellular Encoding of Spatiotemporal Guidance Cues in a Self-Organizing Signaling System for Chemotaxis in Dictyostelium Cells

Even in the absence of guidance cues, chemotactic cells are often spontaneously motile, which should accompany a spontaneous symmetry breaking inside the cells. A shallow chemoattractant gradient can induce these cells to move directionally without much change in cell morphology. As the gradient becomes steeper, the accuracy of chemotaxis increases. It is not clear how the steepness is expressed or encoded internally in the signaling network, which in turn coordinately activates the motile apparatus for chemotaxis. In Dictyostelium cells, self-organizing polarization activities in the signaling network have been reported. In this paper, we conducted a theoretical study of the response of this self-organizing system to guidance cues. Our analyses indicate that self-organizing systems respond sharply to a shallow external gradient by increasing the precision of polarity direction and modulating the frequency of self-polarization. We also show how the precision increase and frequency modulation are achieved. Our results indicate that self-organizing activity, independent of external cues, is the basis for the sensitive and robust response to shallow gradients. Finally, we show that the system can sense the direction of space-time waves of a stimulus, for which Dictyostelium cells exhibit chemotaxis in the developmental process.

Directional sensing of deformed cells under faint gradients

We consider the physical limit of the directional sensing ability of living cells, as in chemotaxis, under a low concentration and shallow chemoattractant gradient. Elliptic cells sense the direction, which is a stochastic variable of a characteristic distribution with peaks at directions not necessarily to the gradient. The peak positions depend on the information of the gradient that cells use to infer the direction and also the shape and orientation of cells. Cells of different shapes may use different inference strategies to increase their directional sensing performance.

Reactive oxygen species as signaling molecules in neutrophil chemotaxis

Neutrophil chemotaxis is a critical component in innate immunity. Recently, using a small-molecule functional screening, we identified NADPH-oxidase-dependent Reactive Oxygen Species (ROS) as key regulators of neutrophil chemotactic migration. Neutrophils depleted of ROS form more frequent multiple pseudopodia and lost their directionality as they migrate up a chemoattractant concentration gradient. Here, we further studied the role of ROS in neutrophil chemotaxis and found that multiple pseudopodia formation induced by NADPH inhibitor diphenyleneiodonium chloride (DPI) was more prominent in relatively shallow chemoattractant gradient. It was reported that, in shallow chemoattractant gradients, new pseudopods are usually generated when existing ones bifurcate. Directional sensing is mediated by maintaining the most accurate existing pseudopod, and destroying pseudopods facing the wrong direction by actin depolymerization. We propose that NADPH-mediated ROS production may be critical for disruption of misoriented pseudopods in chemotaxing neutrophils. Thus, inhibition of ROS production will lead to formation of multiple pseudopodia.

Navigation of Chemotactic Cells by Parallel Signaling to Pseudopod Persistence and Orientation

The mechanism of chemotaxis is one of the most interesting issues in modern cell biology. Recent work shows that shallow chemoattractant gradients do not induce the generation of pseudopods, as has been predicted in many models. This poses the question of how else cells can steer towards chemoattractants. Here we use a new computational algorithm to analyze the extension of pseudopods by Dictyostelium cells. We show that a shallow gradient of cAMP induces a small bias in the direction of pseudopod extension, without significantly affecting parameters such as pseudopod frequency or size. Persistent movement, caused by alternating left/right splitting of existing pseudopodia, amplifies the effects of this bias by up to 5-fold. Known players in chemotactic pathways play contrasting parts in this mechanism; PLA2 and cGMP signal to the cytoskeleton to regulate the splitting process, while PI 3-kinase and soluble guanylyl cyclase mediate the directional bias. The coordinated regulation of pseudopod generation, orientation and persistence by multiple signaling pathways allows eukaryotic cells to detect extremely shallow gradients.

Negative feedback regulation of Rac in leukocytes from mice expressing a constitutively active phosphatidylinositol 3-kinase γ

Polarization of chemotaxing cells depends on positive feedback loops that amplify shallow gradients of chemoattractants into sharp intracellular responses. In particular, reciprocal activation of phosphatidylinositol 3-kinases (PI3Ks) and small GTPases like Rac leads to accumulation, at the leading edge, of the PI3K product phosphatidylinositol 3,4,5-trisphosphate (PIP3). Mice carrying a "knockin" allele of the G protein-coupled receptor (GPCR)-activated PI3Kgamma, encoding a plasma membrane-targeted protein appeared normal, but their leukocytes showed GPCR-uncoupled PIP3 accumulation. In vivo, the mutation increased proliferation and decreased apoptosis, leading to leukocytosis and delayed resolution of inflammation in wound healing. Mutant leukocytes showed significantly impaired directional cell migration in response to chemoattractants. Stimulated mutant macrophages did not polarize PIP3 and showed a shortened Rac activation because of enhanced PI3K-dependent activation of RacGAPs. Together with the finding that chemoattractants stimulate a PIP3-dependent GAP activation in wild-type macrophages, these results identify a molecular mechanism involving PI3K- and RacGAP-dependent negative control of Rac that limits and fine-tunes feedback loops promoting cell polarization and directional motility.

Chemotaxis in shallow gradients is mediated independently of PtdIns 3-kinase by biased choices between random protrusions

Current models of eukaryotic chemotaxis propose that directional sensing causes localized generation of new pseudopods. However, quantitative analysis of pseudopod generation suggests a fundamentally different mechanism for chemotaxis in shallow gradients: first, pseudopods in multiple cell types are usually generated when existing ones bifurcate and are rarely made de novo; second, in Dictyostelium cells in shallow chemoattractant gradients, pseudopods are made at the same rate whether cells are moving up or down gradients. The location and direction of new pseudopods are random within the range allowed by bifurcation and are not oriented by chemoattractants. Thus, pseudopod generation is controlled independently of chemotactic signalling. Third, directional sensing is mediated by maintaining the most accurate existing pseudopod, rather than through the generation of new ones. Finally, the phosphatidylinositol 3-kinase (PI(3)K) inhibitor LY294002 affects the frequency of pseudopod generation, but not the accuracy of selection, suggesting that PI(3)K regulates the underlying mechanism of cell movement, rather than control of direction.

Is there a pilot in a pseudopod?

The concept of pilot pseudopodia is reconsidered 30 years after its inauguration (Gerisch, G., Hülser, D., Malchow, D., Wick, U., 1975. Cell communication by periodic cyclic-AMP pulses. Phil. Trans. R. Soc. Lond. B 272, 181–192). The original hypothesis stated that protruding pseudopodia serve as dynamic sensory organelles that aid a cell in perceiving variations of chemoattractant concentration and, consequently, in navigation during chemotaxis. This influential idea is reevaluated in the light of recent findings about the mechanisms governing chemotactic cell motility, morphology and dynamics of pseudopodia, and about molecular constituents and regulators of pseudopod extension and retraction. It is proposed that stimulation by a chemoattractant modulates speed of pseudopod protrusion and thereby increases cell elongation. Elongation further enhances chemotactic sensitivity of the cell to shallow chemoattractant gradients, reinforces cell polarization, and finally leads to suppression of lateral pseudopodia and continuation of cell migration in the gradient direction.

Diffusion-limited phase separation in eukaryotic chemotaxis

The ability of cells to sense spatial gradients of chemoattractant factors governs the development of complex eukaryotic organisms. Cells exposed to shallow chemoattractant gradients respond with strong accumulation of the enzyme phosphatidylinositol 3-kinase (PI3K) and its D3-phosphoinositide product (PIP(3)) on the plasma membrane side exposed to the highest chemoattractant concentration, whereas PIP(3)-degrading enzyme PTEN and its product PIP(2) localize in a complementary pattern. Such an early symmetry-breaking event is a mandatory step for directed cell movement elicited by chemoattractants, but its physical origin is still mysterious. Here, we propose that directional sensing is the consequence of a phase-ordering process mediated by phosphoinositide diffusion and driven by the distribution of chemotactic signal. By studying a realistic reaction-diffusion lattice model that describes PI3K and PTEN enzymatic activity, recruitment to the plasma membrane, and diffusion of their phosphoinositide products, we show that the effective enzyme-enzyme interaction induced by catalysis and diffusion introduces an instability of the system toward phase separation for realistic values of physical parameters. In this framework, large reversible amplification of shallow chemotactic gradients, selective localization of chemical factors, macroscopic response timescales, and spontaneous polarization arise naturally. The model is robust with respect to order-of-magnitude variations of the parameters.

Receptor Occupancy on an Ellipsoidal Cell in the Presence of a Point Source of a Chemoattractant

Diffusion of a chemoattractant from a micropipet is routinely used to examine the different aspects of a cell's chemotactic response. To quantify the effect of cell elongation on chemotactic sensitivity in the micropipet assay, the chemoattractant concentration at the cell plasma membrane was determined by solving the equation for diffusion from a point source in the presence of a prolate ellipsoid of varying eccentricity. The results show that cell elongation can significantly increase the difference in receptor occupancy between near and far cell ends and thereby enhance the sensitivity of chemotactic cells to shallow chemoattractant gradients.

Signaling mechanisms for regulation of chemotaxis

Chemotaxis is a fascinating biological process, through which a cell migrates along a shallow chemoattractant gradient that is less than 5% difference between the anterior and posterior of the cell. Chemotaxis is composed of two independent, but interrelated processes-motility and directionality, both of which are regulated by extracellular stimuli, chemoattractants. In this mini-review, recent progresses in the understanding of the regulation of leukocyte chemotaxis by chemoattractant signaling are reviewed.

Two Complementary, Local Excitation, Global Inhibition Mechanisms Acting in Parallel Can Explain the Chemoattractant-Induced Regulation of PI(3,4,5)P 3 Response in Dictyostelium Cells

Chemotaxing cells, such as Dictyostelium and mammalian neutrophils, sense shallow chemoattractant gradients and respond with highly polarized changes in cell morphology and motility. Uniform chemoattractant stimulation induces the transient translocations of several downstream signaling components, including phosphoinositide 3-kinase (PI3K), tensin homology protein (PTEN), and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3). In contrast, static spatial chemoattractant gradients elicit the persistent, amplified localization of these molecules. We have proposed a model in which the response to chemoattractant is regulated by a balance of a local excitation and a global inhibition, both of which are controlled by receptor occupancy. This model can account for both the transient and spatial responses to chemoattractants, but alone does not amplify the external gradient. In this article, we develop a model in which parallel local excitation, global inhibition mechanisms control the membrane binding of PI3K and PTEN. Together, the action of these enzymes induces an amplified PI(3,4,5)P 3 response that agrees quantitatively with experimentally obtained plekstrin homology-green fluorescent protein distributions in latrunculin-treated cells. We compare the model’s performance with that of several mutants in which one or both of the enzymes are disrupted. The model accounts for the observed response to multiple, simultaneous chemoattractant cues and can recreate the cellular response to combinations of temporal and spatial stimuli. Finally, we use the model to predict the response of a cell where only a fraction is stimulated by a saturating dose of chemoattractant.

Stochastic modelling for gradient sensing by chemotactic cells

Chemotaxis, the process by which cells move toward attractant molecules, operates in a range of biological processes including immunity, neuronal patterning, and morphogenesis. Dictyostelium discoideum cells display a strong chemotactic response to cyclic adenosine 3',5'-monophosphate (cAMP), which binds to a cell surface receptor. Each Dictyostelium has ca. 80000 cAMP receptors, and can transduce shallow spatial chemoattractant gradients into strongly localized intracellular responses in spite of large statistical fluctuation of receptor occupancy even in the case of very low cAMP concentration. In this study, we develop a stochastic model for gradient sensing by chemotactic cells. We simulate the binding of cAMP molecules to receptors by a Monte-Carlo method in order to account for statistical fluctuation of receptor occupancy and treat intracellular signal processing by a diffusion–translocation model, which includes the production of second-messenger molecules and positive feedback mechanisms mediated by effector molecules. Our simulation results show that the fluctuation of second-messenger concentration is much smaller than that of receptor occupancy, and that a shallow chemoattractant gradient are transduced into a large second-messenger concentration gradient through nonlinear signal amplification.

Focus Issue: Building Signaling Connections

Focus Issue: Building Signaling Connections

Building a Case for Signaling

T his week's coordinated special issues of Science and Science 's Signal Transduction Knowledge Environment (STKE) ( ) feature additions to the signaling pathway database at STKE: the Connections Maps.[*][1] This database organizes information on the signaling circuitry that regulates cellular function. The Viewpoints in this issue summarize current understanding of how these pathways regulate diverse functions in health and disease. Biochemical signaling pathways interact to form a complicated network that becomes evident when viewing the Connections Maps pathways. “Pathway” units are used for convenience, but new navigational tools allow users to “walk” from one component to another, exploring how the same component works in multiple pathways. “Interpathway connections” show branch points where one pathway influences another [see the Editorial Guide at STKE ([www.stke.org/cgi/content/full/sigtrans;2003/186/eg8][2])]. Viewpoints from Barton and Medzhitov (p. 1524) and Kelly-Welch et al. (p. [1527][3]) discuss signaling by Toll-like receptors and interleukins (ILs). The Toll-like receptors enable the body to detect the presence of microbial invaders and initiate processes that defend against such an attack. Signals mediated by IL-4 and IL-13 present potential therapeutic targets for treatment of asthma and allergies. Xiang and Kobilka (p. 1530) describe signaling by adrenoceptors, which control normal heart function and can contribute to heart failure. Vaudry et al. (p. [1532][4]) address multiple pathways that contribute to life-or-death decisions in cerebellar neurons. Important early in life for proper developmental patterning, these pathways may later malfunction in neurodegenerative diseases. Similar circuits can be constructed from different components, as Van Gelder (p. [1534][5]) describes for pathways that make up biological clocks. Some surprises come from the huge family of receptors that couple to heterotrimeric guanine nucleotide-binding proteins (G proteins). Kimmel and Parent (p. 1525) describe a receptor in the social ameoba Dictyostelium that signals through a G protein but also triggers G protein-independent signals. This model organism provides a system for understanding how cells orient themselves in very shallow gradients of chemoattractants. Wang and Malbon (p. 1529) describe a receptor that has structural characteristics of a G protein-coupled receptor but is best understood to signal by a different mechanism. The authors summarize evidence that these receptors (“Frizzleds”) may also use a traditional G protein activation mechanism. We hope the Viewpoints lead you to explore the Connections Maps at Science's STKE. We anticipate that a comprehensive signaling database will enable analysis not previously possible with information stored in the text of traditional research publications. [1]: #fn-1 [2]: http://www.stke.org/cgi/content/full/sigtrans;2003/186/eg8 [3]: /lookup/doi/10.1126/science.1085458 [4]: /lookup/doi/10.1126/science.1085260 [5]: /lookup/doi/10.1126/science.1085446